Abstract Short INterspersed Elements (SINEs) comprise approximately 13% of human genomic DNA. The vast majority of SINEs have been rendered immobile by mutational processes, but some (e.g., human Alu elements) can still mobilize throughout the genome by a process termed retrotransposition. SINEs do not encode proteins; thus, they are classified as `non-autonomous' retrotransposons. Previous studies, including our preliminary data, demonstrate that a protein encoded by an autonomous Long INterspersed Element-1 (LINE-1 or L1) retrotransposon (L1 ORF2p) can be co-opted by Alu to promote its retrotransposition in trans. To date, 76 independent germline Alu retrotransposition events have been implicated as the cause of human diseases, including cancer. We recently demonstrated that L1 ORF2p binds to the poly(A) sequence present at the 3' end of LINE-1 and Alu RNAs to promote retrotransposition. These and other data lead us to hypothesize that a cellular factor(s) associates with Alu RNA to transport it to the ribosome, and this localization allows the Alu poly(A) tract to compete with the L1 poly(A) tail for the binding of nascent L1 ORF2p. We also found that HeLa cell isolates (i.e., strains) differ in their capacity to support Alu retrotransposition. One strain, HeLa-HA, is permissive and supports both human LINE-1 and Alu retrotransposition. Another strain, HeLa- JVM, is non-permissive and supports LINE-1, but not Alu, retrotransposition. These data suggest: 1) HeLa-JVM cells express a dominant suppressor(s) of Alu retrotransposition; or 2) HeLa-JVM cells lack the expression of a host factor(s) required for Alu retrotransposition. This R21 proposal builds upon our previous studies and preliminary data and will use permissive and non-permissive HeLa strains in conjunction with molecular biological, genomics, biochemical, genetic complementation and genome-engineering approaches to identify host factor(s) that are required for Alu retrotransposition. Our strategy meets the `high-risk/high-payoff' requirement of an R21 and represents a strategy to identify a host defense pathway that restricts Alu retrotransposition in human cancer cell lines and, perhaps, human cancers.